Forty percent of all deaths in the United States result from cardiovascular disease at an estimated annual cost of over 400 billion dollars. Endogenous hormones called natriuretic peptides combat cardiovascular disease. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are released from the heart in response to elevated blood pressure. They bind natriuretic peptide receptor-A (NPR-A), which decreases blood pressure and inhibits cardiac hypertrophy by synthesizing the intracellular signaling molecule cGMP. C-type natriuretic peptide (CNP) is a paracrine factor that stimulates long bone growth and inhibits vasorelaxation by activating natriuretic peptide receptor-B (NPR-B). The guanylyl cyclase activity of NPR-A and NPR-B is controlled not only by natriuretic peptide binding but also by enzymes that regulate the amount of phosphate covalently attached to highly conserved serines and threonine residues within their intracellular domains. Importantly, phosphorylation is required for receptor activation and dephosphorylation mediates receptor inactivation. Our long-term goal is to understand the molecular basis of natriuretic peptide receptor control in health and disease. The objective of this application is to identify the kinase that phosphorylates NPR-A. Our central hypothesis is that receptor phosphorylation is required for ligand-dependent activation of NPR-A, and therefore, is essential for cardiac natriuretic peptide signal transduction. Each specific aim describes a unique approach to identify the natriuretic peptide receptor kinase. We have shown that a glutathione-S-transferase (GST) protein fused to the intracellular domain of NPR-A stably associates with a molecule that phosphorylates NPR-A. The first Specific Aim of this application is to identify the proteins that associate with GST-NPR-A by MALDI-TOF mass spectrometry. The second Specific Aim is to perform a siRNA screen against all known human protein kinases using reductions in ANP-dependent cGMP elevations in Hela cells as an indicator of reduced NPR-A phosphorylation. The third Specific Aim uses an in-gel kinase assay coupled with mass spectrometry to identify the NPR-A kinase. Finally, the fourth Specific Aim takes advantage of a novel bifunctional ATP analog to cross- link NPR-A to its cognate kinase. The identification of the NPR-A kinase is anticipated to provide the framework for future therapeutic targeting of this molecule for treatment of hypertension, congestive heart disease and skeletal disorders. PUBLIC HEALTH RELEVANCE: Natriuretic peptides inhibit blood pressure and stimulate long bone growth in humans. Unfortunately, one of the key regulators of this endocrine system, the natriuretic peptide receptor kinase, is unknown. The goal of this grant is to identify this critical molecule so that it can be targeted by novel drugs designed to treat cardiovascular and skeletal diseases.